Zeyu Wu1,2, Ke Wang2, Zhenyu Yang1,2, Laura E Pascal2, Joel B Nelson2,3, Keita Takubo4, Peter Wipf3,4,5, Zhou Wang2,3,5,6. 1. Department of Urology, Xiangya School of Medicine, Xiangya School of Medicine, Central South University, Changsha, China. 2. Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. 3. UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. 4. Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania. 5. Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. 6. Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Abstract
BACKGROUND: Castration-resistant prostate cancer can develop resistance to enzalutamide because of androgen receptor (AR) point mutations, AR overexpression, constitutively active AR splice variants, and/or elevated intratumoral androgen synthesis. The point mutation ARF876L was reported to be stimulated, instead of inhibited, by enzalutamide, thus contributing to enzalutamide resistance. We have recently developed JJ-450 as a novel AR antagonist with the potential to treat enzalutamide-resistant castration-resistant prostate cancer (CRPC). METHODS: We employed several assays to determine the impact of JJ-450 and enzalutamide on prostate cancer cell lines expressing green fluorescent protein (GFP)-ARF876L . These assays include a prostate-specific antigen enhancer/promoter-based luciferase assay to determine AR transcriptional activity, a quantitative real-time polymerase chain reaction assay, and Western blot analysis to detect expression of AR-target genes at the messenger RNA and protein level, fluorescence microscopy to show AR subcellular localization, and a 5-bromo-2'-deoxyuridine assay to measure prostate cancer cell proliferation. RESULTS: As expected, enzalutamide inhibited wild-type (WT) AR but not ARF876L transcriptional activity in the luciferase assay. In contrast, JJ-450 inhibited both WT-AR and ARF876L transcriptional activity to a similar extent. Also, enzalutamide retarded androgen-induced nuclear import of GFP-AR, but not GFP-ARF876L , whereas JJ-450 retarded nuclear import of both GFP-AR and GFP-ARF876L . To further evaluate JJ-450 inhibition of ARF876L , we stably transfected C4-2 cells separately with GFP-AR or GFP-ARF876L . Enzalutamide inhibited endogenous AR-target gene expression in C4-2-GFP-ARWT , but not in the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited AR-target gene expression in both C4-2 sublines. More importantly, enzalutamide inhibited proliferation of C4-2-GFP-ARWT , but not of the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited proliferation of both C4-2 sublines. CONCLUSION: JJ-450 inhibits enzalutamide-resistant ARF876L mutant nuclear translocation and function. Our findings suggest that JJ-450 and its analogs should be further developed to provide a potential new approach for the treatment of enzalutamide-resistant CRPC.
BACKGROUND: Castration-resistant prostate cancer can develop resistance to enzalutamide because of androgen receptor (AR) point mutations, AR overexpression, constitutively active AR splice variants, and/or elevated intratumoral androgen synthesis. The point mutation ARF876L was reported to be stimulated, instead of inhibited, by enzalutamide, thus contributing to enzalutamide resistance. We have recently developed JJ-450 as a novel AR antagonist with the potential to treat enzalutamide-resistant castration-resistant prostate cancer (CRPC). METHODS: We employed several assays to determine the impact of JJ-450 and enzalutamide on prostate cancer cell lines expressing green fluorescent protein (GFP)-ARF876L . These assays include a prostate-specific antigen enhancer/promoter-based luciferase assay to determine AR transcriptional activity, a quantitative real-time polymerase chain reaction assay, and Western blot analysis to detect expression of AR-target genes at the messenger RNA and protein level, fluorescence microscopy to show AR subcellular localization, and a 5-bromo-2'-deoxyuridine assay to measure prostate cancer cell proliferation. RESULTS: As expected, enzalutamide inhibited wild-type (WT) AR but not ARF876L transcriptional activity in the luciferase assay. In contrast, JJ-450 inhibited both WT-AR and ARF876L transcriptional activity to a similar extent. Also, enzalutamide retarded androgen-induced nuclear import of GFP-AR, but not GFP-ARF876L , whereas JJ-450 retarded nuclear import of both GFP-AR and GFP-ARF876L . To further evaluate JJ-450 inhibition of ARF876L , we stably transfected C4-2 cells separately with GFP-AR or GFP-ARF876L . Enzalutamide inhibited endogenous AR-target gene expression in C4-2-GFP-ARWT , but not in the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited AR-target gene expression in both C4-2 sublines. More importantly, enzalutamide inhibited proliferation of C4-2-GFP-ARWT , but not of the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited proliferation of both C4-2 sublines. CONCLUSION:JJ-450 inhibits enzalutamide-resistant ARF876L mutant nuclear translocation and function. Our findings suggest that JJ-450 and its analogs should be further developed to provide a potential new approach for the treatment of enzalutamide-resistant CRPC.
Authors: Peter S Nelson; Nigel Clegg; Hugh Arnold; Camari Ferguson; Michael Bonham; James White; Leroy Hood; Biaoyang Lin Journal: Proc Natl Acad Sci U S A Date: 2002-08-16 Impact factor: 11.205
Authors: Paul Toren; Soojin Kim; Thomas Cordonnier; Claire Crafter; Barry R Davies; Ladan Fazli; Martin E Gleave; Amina Zoubeidi Journal: Eur Urol Date: 2014-08-20 Impact factor: 20.096
Authors: Zhenyu Yang; Dan Wang; James K Johnson; Laura E Pascal; Keita Takubo; Raghunandan Avula; Anish Bhaswanth Chakka; Jianhua Zhou; Wei Chen; Mingming Zhong; Qiong Song; Hui Ding; Zeyu Wu; Uma R Chandran; Taber S Maskrey; Joel B Nelson; Peter Wipf; Zhou Wang Journal: Mol Cancer Ther Date: 2019-09-25 Impact factor: 6.261
Authors: Charles J Ryan; Matthew R Smith; Johann S de Bono; Arturo Molina; Christopher J Logothetis; Paul de Souza; Karim Fizazi; Paul Mainwaring; Josep M Piulats; Siobhan Ng; Joan Carles; Peter F A Mulders; Ethan Basch; Eric J Small; Fred Saad; Dirk Schrijvers; Hendrik Van Poppel; Som D Mukherjee; Henrik Suttmann; Winald R Gerritsen; Thomas W Flaig; Daniel J George; Evan Y Yu; Eleni Efstathiou; Allan Pantuck; Eric Winquist; Celestia S Higano; Mary-Ellen Taplin; Youn Park; Thian Kheoh; Thomas Griffin; Howard I Scher; Dana E Rathkopf Journal: N Engl J Med Date: 2012-12-10 Impact factor: 91.245
Authors: James D Joseph; Nhin Lu; Jing Qian; John Sensintaffar; Gang Shao; Dan Brigham; Michael Moon; Edna Chow Maneval; Isan Chen; Beatrice Darimont; Jeffrey H Hager Journal: Cancer Discov Date: 2013-06-18 Impact factor: 39.397
Authors: Khalid Z Masoodi; Kurtis Eisermann; Zhenyu Yang; Javid A Dar; Laura E Pascal; Minh Nguyen; Katherine O'Malley; Erica Parrinello; Firuz G Feturi; Alex N Kenefake; Joel B Nelson; Paul A Johnston; Peter Wipf; Zhou Wang Journal: Endocrinology Date: 2017-10-01 Impact factor: 4.736
Authors: Melanie Mediavilla-Varela; Fabio J Pacheco; Frankis Almaguel; Jossymar Perez; Eva Sahakian; Tracy R Daniels; Lai Sum Leoh; Amelia Padilla; Nathan R Wall; Michael B Lilly; Marino De Leon; Carlos A Casiano Journal: Mol Cancer Date: 2009-08-28 Impact factor: 27.401